Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2000-10-16
2002-10-22
LeGuyader, John L. (Department: 1635)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S006120, C435S091100, C435S325000, C435S366000, C536S023100, C536S024310, C536S024330, C536S024500
Reexamination Certificate
active
06468795
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of Apaf-1. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding Apaf-1. Such compounds have been shown to modulate the expression of Apaf-1.
BACKGROUND OF THE INVENTION
Apoptosis, or programmed cell death, is a naturally occurring process that has been strongly conserved during evolution to prevent uncontrolled cell proliferation. This form of cell suicide plays a crucial role in ensuring the development and maintenance of multicellular organisms by eliminating superfluous or unwanted cells. However, if this process goes awry becoming overstimulated, cell loss and degenerative disorders including neurological disorders such as Alzheimers, Parkinsons, ALS, retinitis pigmentosa and blood cell disorders can result. Stimuli which can trigger apoptosis include growth factors such as tumor necrosis factor (TNF), Fas and transforming growth factor beta (TGF&bgr;), neurotransmitters, growth factor withdrawal, loss of extracellular matrix attachment and extreme fluctuations in intracellular calcium levels (Afford and Randhawa,
Mol. Pathol
., 2000, 53, 55-63).
Alternatively, insufficient apoptosis, triggered by growth factors, extracellular matrix changes, CD40 ligand, viral gene products neutral amino acids, zinc, estrogen and androgens, can contribute to the development of cancer, autoimmune disorders and viral infections (Afford and Randhawa,
Mol. Pathol
., 2000, 53, 55-63). Consequently, apoptosis is regulated under normal circumstances by the interaction of gene products that either induce or inhibit cell death and several gene products which modulate the apoptotic process have now been identified.
Apoptotic protease activating factor 1 (also known as Apaf-1) is the human homolog of the
C. elegans
gene, ced-4, originally purified and cloned from HeLa cell cytosol (Zou et al.,
Cell
, 1997, 90, 405-413). Disclosed in the PCT Publication WO 98/55615 are the protein and nucleic acid sequences of Apaf-1 as are antibodies to the protein, host cells which express a vector encoding the nucleic acid sequence of Apaf-1, transgenic and knock-out animals and methods to detect Apaf-1 modulating compounds. Generally disclosed is an isolated nucleic acid comprising a nucleotide which would hybridize to Apaf-1 under stringent conditions (Zou et al., 1998).
Characterization of the protein has revealed that Apaf-1 contains a domain structure with defined regions of homology to existing proteins including WD repeats, and caspase-related domains (Zou et al.,
Cell
, 1997, 90, 405-413). Tissue localization studies have demonstrated that Apaf-1 can be found in most tissues (Zou et al.,
Cell
, 1997, 90, 405-413), however there is debate on the presence of Apaf-1 in skeletal muscle (Burgess et al.,
Cell Death and Differentiation
, 1999, 6, 256-261). Within the cell, Apaf-1 is localized to the cytosol (Hausmann et al.,
Journal of Cell Biology
, 2000, 149, 623-634) and at least three variants of the protein have been identified to date. Hahn et al. describe three novel forms of Apaf-1 isolated from six lymphoma cell lines, three non-lymphoid tumor cell lines, peripheral blood lymphocytes, and in tissues from heart, kidney and liver (Hahn et al.,
Biochemical and Biophysical Research Communications
, 1999, 261, 746-749).
Others have also identified alternate forms of Apaf-1 resulting from differential splicing events (Benedict et al.,
Journal of Biological Chemistry
, 2000, 275, 8461-8468; Hu et al.,
The Embo Journal
, 1999, 18, 3586-3595). Characterization of these variants demonstrated that longer forms of the protein containing an extra WD repeat were required to activate the apoptotic cascade orchestrated by caspases (Benedict et al.,
Journal of Biological Chemistry
, 2000, 275, 8461-8468). Disclosed in the PCT publication WO 99/65937 are the nucleic acid sequence of Apaf-1 including truncated variants of Apaf-1 which may oligomerize with members of the caspase family as are host cells which express a vector encoding the nucleic acid sequence of Apaf-1, and methods to detect inhibitors of apoptosis and Apaf-1 modulating compounds. Generally disclosed is an isolated nucleic acid comprising a nucleotide which would hybridize to Apaf-1 under stringent conditions (Alnemri, 1999).
Since its isolation, Apaf-1 has been shown to play a critical role in the regulation of apoptosis through multiple signaling pathways reviewed in (Cecconi,
Cell Death and Differentiation
, 1999, 6, 1087-1098). Primarily Apaf-1 acts through cytochrome c-mediated caspase activation (Saleh et al.,
Journal of Biological Chemistry
, 1999, 274, 17941-17945). Upon release of cytochrome c from the mitochondria and in the presence of DATP (deoxy ATP), Apaf-1 oligomerizes and forms a complex with procaspase-9 (Cain et al.,
Journal of Biological Chemistry
, 2000, 275, 6067-6070; Zou et al.,
Journal of Biological Chemistry
, 1999, 274, 11549-11556). In turn, procaspase-9 is cleaved into its active form, leading to the activation of caspase-3, the major caspase in the apoptotic pathway. Overexpression of Apaf-1 has been shown to increase sensitivity of human myeloid leukemia HL-60 cells to the apoptosis-inducing agents paclitaxel and etoposide (Perkins et al.,
Cancer Research
, 1998, 58, 4561-4566).
Although Apaf-1 has been localized to chromosome 12q23, a region recurrently deleted in male germ cell tumors, genetic analysis has shown that Apaf-1 is not a tumor suppressor gene candidate (Bala et al.,
Genes, Chromosomes
&
Cancer
, 2000, 28, 258-268; Kim et al.,
Cytogenet. Cell Genet
., 1999, 87, 252-253). Expression of Apaf-1, nonetheless has been associated with several types of cancers. Frameshift mutations in the Apaf-1 gene have been suggested to provide a survival advantage to some tumor types (Yamamoto et al.,
Cell Death and Differentiation
, 2000, 7, 238-239) and mouse embryonic cells lacking Apaf-1 and caspase-9 were shown to be resistant to apoptotic stimuli (Soengas et al.,
Science
, 1999, 284, 156-159). The majority of homozygous knockout mice lacking Apaf-1 die early in embryogenesis and show reduced apoptosis leading to many morphological abnormalities. In the mice reaching maturity, the adult heterozygote males exhibit infertility (Cecconi et al.,
Cell
, 1998, 94, 727-737; Honarpour et al.,
Cevelopmental Biology
, 2000, 218, 248-258).
Collectively, these data suggest that modulation of Apaf-1 would render opportunity to treat patients with various cancers and deregulated apoptotic pathologic conditions.
Strategies aimed at modulating Apaf-1 function have involved the use of antibodies and gene knockouts in mice. However, these strategies are untested as therapeutic protocols and consequently there remains a long felt need for agents capable of effectively inhibiting Apaf-1 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of Apaf-1.
The present invention provides compositions and methods for modulating Apaf-1 expression, including modulation of the truncated forms, including splice variants of Apaf-1.
SUMMARY OF THE INVENTION
The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding Apaf-1, and which modulate the expression of Apaf-1. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of Apaf-1 in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of Apaf-1 by administering a therapeutically or pro
ISIS Pharmaceuticals Inc.
LeGuyader John L.
Licata & Tyrrell P.C.
Schmidt M
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